Only recently has it been possible to transfer heterologous (i.e., foreign) nucleic acid sequences, such as control elements or gene coding regions, into cells, let alone into animals. Investigators have been testing a variety of delivery vehicles to both introduce and maintain a gene in the cells of an animal, such as a human, to correct, for example, inborn errors of metabolism.
The majority of gene transfer vehicles tested have included virus vectors that integrate into the host genome. Although integration of a vector may be desirable to achieve stable gene transfer, there is a danger that the vector may integrate at a position that could harm the animal. For example, one concern about the use of retroviruses and adeno-associated viruses is that, since integration of the virus vector typically cannot be controlled, there is a potential danger that, upon integration, the vector will activate the expression of an undesired gene located near it, such as an oncogene.
Most research and clinical efforts to date have used retroviruses as drug delivery vehicles, particularly to deliver genes to hematopoietic cells; see, for example, Beutler et al., pp. 857-860, 1990, Exp. Hematol., Vol. 18; Kopchick et al., U.S. Pat. No. 4,828,987, issued May 9, 1989; Wagner et al., U.S. Pat. No. 5,032,407, issued Jul. 16, 1991; Goldsmith et al., PCT International Publication No. WO 90/07936. However, in many cases, expression of recombinant retrovirus vectors in undifferentiated cells has been suppressed; see, for example, Beutler, ibid. In addition, retroviruses are quite labile, grow to relatively low titers (particularly recombinant retroviruses), are difficult to handle without significant infectivity loss, and exhibit a limited host range.
Adeno-associated viruses are also being evaluated as possible gene delivery vehicles. For a review, see Muzyczka, pp. 97-129, 1992, Current Topics in Microbiology and Immunology, Vol. 158; also see, for example Carter et al., U.S. Pat. No. 4,797,368, issued Jan. 10, 1989; Chatterjee et al., PCT International Publication No. WO 91/18088. Adeno-associated viruses are a genus of parvoviruses that integrate into the host genome and require either adenovirus or a herpes virus to replicate. Little is known about the long-term effects of these viruses on a host, such as the effects of integration. In addition, due to their dependence on other viruses to replicate, adeno-associated viruses are difficult to produce.
Researchers are also assessing the usefulness of viruses that remain essentially autonomous (i.e., do not appear to integrate to a significant amount) to effect stable gene transfer. Examples of such viruses include adenovirus, bovine papilloma virus, and various herpes viruses. See, for example, Rosenfeld et al., pp. 431-434, 1991, Science, Vol 252; Stratford-Perricaudet et al., pp. 241-256, 1990, Human Gene Therapy, Vol. 1; Tani et al., pp. 1274-1280, 1989, Blood, Vol. 74; Beutler et al., ibid.; Howley et al., U.S. Pat. No. 4,419,446, issued Dec. 6, 1983; Salser et al., U.S. Pat. No. 4,497,796, issued Feb. 5, 1985. There are, however, a variety of safety concerns about a number of such viruses, and the host range of at least some of these viruses is limited. In addition, production of such viruses is complicated by the number of virus proteins required for virus vector amplification and, if desired, encapsidation. There is also a concern that replication-defective vectors may recombine with helper virus vectors to create infectious viruses.
Additional methods to deliver genes, including direct administration of naked DNA or RNA molecules and attachment of nucleic acids to carriers, such as liposomes, are being developed. See, for example, Gould-Fogerite et al., pp. 429-438, 1989 Gene, Vol. 84; Brigham, PCT International Publication No. WO 91/06309.
Despite the progress made to date, there is still a need for gene delivery vehicles that target appropriate cell types when administered in vivo (i.e., directly administered to the animal rather than transferring genes to cells outside the body) and that elicit gene expression in desired cell types. There is also a need for a gene delivery vehicle that effects transient gene therapy, particularly in the treatment of a number of diseases, such as cancer and diseases caused by infectious agents. For these diseases, it may be desirable to target a gene encoding a cytotoxin to affected cells.
Certain toxin-encoding genes, such as the gene for diphtheria toxin, have been shown to be selectively expressed in certain desired cell types, but in general, such targeting has been achieved by operatively linking the gene to a transcription control element that is substantially only activated in the desired cell type. See, for example, Maxwell et al., pp. 4660-4664, 1986, Cancer Res., Vol. 46; Maxwell et al., pp. 4299-4304, 1991, Cancer Res., Vol. 51; Palmiter et al., pp. 435-443, 1987, Cell, Vol. 50; Breitman et al., pp. 1563-1565, 1987, Science, Vol. 238; Breitman et al., pp. 474-479, 1990, Mol. Cell. Biol., Vol. 10; Harrison et al., pp. 53-60, 1991, Human Gene Therapy, Vol. 2. Such selective expression of genes transferred to a cell by a vector has been tested in cell culture and transgenic animals. There still remains, however, a need to develop vehicles that selectively deliver such vectors to desired cell types, particularly for human applications. There is also a need to develop gene delivery vehicles that are easier to produce than vehicles based on, for example, adeno-associated viruses, adenoviruses, or retroviruses.
Autonomous parvoviruses are small DNA viruses that replicate autonomously in rapidly dividing cells. The genomes of autonomous parvoviruses apparently do not integrate, at least not at a detectable level. Autonomous parvovirus genomes are single-stranded DNA molecules about 5 kilobases (kb) in size. The genomes are organized such that the NS gene encoding the nonstructural polypeptides NS1 and NS2 is located on the left side of the genome and the VP gene encoding the structural polypeptides required for capsid formation are on the right side of the genome. Expression of the nonstructural polypeptides is controlled by a transcription control sequence called P4 in most parvoviruses, which is located at about map unit position 4 of the genome (assuming the entire genome is 100 map units and numbering is from left to right). Expression of the structural polypeptides is controlled by a transcription control sequence called P38, P39 or P40 in most parvoviruses, which is located at about map unit position 38 to about 40, depending on the autonomous parvovirus. NS1 serves as a trans-activator of the latter transcription control sequence. NS1 is also essential for virus replication and appears to be the primary mediator of parvovirus cytotoxicity, particularly against tumor cells. Autonomous parvovirus genomes also have inverted repeat sequences (i.e., palindromes) at each end which contain essential signals for replication and encapsidation of the virus. There have been several studies on the mechanistics of autonomous parvovirus replication, gene expression, encapsidation, and cytotoxicity. See, for example, Sinkovics, pp. 1281-1290, 1989, Anticancer Res., Vol 9. To the inventors' knowledge, however, autonomous parvoviruses have not been used as gene delivery vehicles prior to the present invention.